CN1091746A - The synthetic peptide of derived from vitronectin and the pharmaceutical composition that contains these synthetic peptides - Google Patents
The synthetic peptide of derived from vitronectin and the pharmaceutical composition that contains these synthetic peptides Download PDFInfo
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- CN1091746A CN1091746A CN93116860A CN93116860A CN1091746A CN 1091746 A CN1091746 A CN 1091746A CN 93116860 A CN93116860 A CN 93116860A CN 93116860 A CN93116860 A CN 93116860A CN 1091746 A CN1091746 A CN 1091746A
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C07—ORGANIC CHEMISTRY
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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Abstract
The present invention relates to tie up out the K of Na Ting (Witronectin) molecule
348-A
380The sequence deutero-synthesizes peptide.Such peptide is regulated the biological activity of plasminogen incitant inhibitor-1 (PAI-1), and be suitable for use as the active ingredient of pharmaceutical composition, this pharmaceutical composition be used for the treatment of disease for example hemorrhagic diseases, Acute Myocardial Infarction, deep-vein thrombosis formation, pulmonary infarction, disseminated inravascular coagulation, tumour cell intrusion and migration, inflammation, hepatopathy, bacillary blood infection, gestosis and with the regulation and control of vasculogenesis or neurotization or the excessive relevant pathological symptom of proteolysis of tpA mediation.
Description
The present invention relates to contain the pharmaceutical composition of tieing up out the synthetic peptide of Na Ting (vitronectin) deutero-, tie up out the biological activity that booth can be regulated plasminogen incitant inhibitor-1(PAI-1), the invention still further relates to some this type of new synthetic peptide.
Hemostasis stops blood effusive process from injured blood vessel, is to be feature with the activity that combines with blood vessel, thrombocyte and blood plasma factor and restrict blood tube wall injured area thrombocyte and scleroproein accumulative balancing.
Hemagglutination has formed the principal element of hemostasis sealing-fibrin clot.By to external diffusion and fix blood platelet embolism, fibrin clot increases to the required size of hemostasis sealing.
Generally have regulation mechanism in case Trostin M reacts, in a single day this reaction is excited, if do not suppressed, then can produce pathological symptom, forms or disseminated inravascular coagulation (DIC) as local thrombus.These regulation mechanisms comprise removes the activated thrombin in the cell, the blood of the liver of particularly flowing through, and the inner neutralization of blood self zymoplasm that is activated and the blood coagulation cofactor mechanism that is activated.
Along with fibrinous deposition, fibrinolytic system is activated.By solution fibrin, this system assists the unobstructed of the injured lumen of vessels of maintenance.Repairing injured vessel wall in required a couple of days, fibrinous deposition and dissolving keep balance and reproduce the hemostasis sealing.
When Fibrinogen when zymoplasm is converted into scleroproein, plasminogen, a kind of inert blood plasma enzyme precursor is by its natural incitant, as organize plasminogen incitant (tPA) to activate, and be converted into effective protein proteins lytic enzyme-blood plasma enzyme, it can be soluble fragments (being called fibrin degradation product (FDP)) with fibrin degradation, and these fragments can be brought into circulation.
Stop several factors of excessive scleroproein hydrolysis to comprise that plasminogen and scleroproein rather than Fibrinogen bonded avidity increase, and when organizing the plasminogen incitant to combine with scleroproein, the ability that it activates plasminogen increase.The main inhibitor of scleroproein hydrolysis is a plasminogen incitant inhibitor-1(PAI-1).Further, blood plasma contains a kind of α of being called
2The proteinase inhibitor of-anti-blood plasma enzyme, this inhibitor can make the blood plasma enzyme deactivation that prevents that fibrin clot from forming rapidly.
In order to stop the effect of blood plasma enzyme, only pass through α
2It is not enough (Collen, 1976) that-anti-blood plasma enzyme makes the excessive blood plasma enzyme deactivation that has formed.Prevent that plasminogen from further producing the blood plasma enzyme also is necessary.This can realize the inhibition of plasminogen incitant by utilization PAI-1, shows PAI-1 and ties up out that booth and combine (Loskutoff et al., 1988; Sprengers and Kluft, 1987), so, PAI-1 has become with its activity form stable existence (Declerck et al., 1988 in blood circulation and extracellular matrix (ECM); Salonen et al., 1989; Mimuro and Loskutoff, 1989; Preissner et al., 1990).
Vitronectir is found as the cellular invasion factor at first, think then that now it is a kind of multi-functional adjusting albumen, it is included in the process of various extracellulars, as the adhering to and spread of normal cell and oncocyte, and plays a role in complement and coagulation pathway.In blood circulation, tieing up out Na Ting has two kinds of molecular form: strand 75KDa polypeptide (V
75) and otch polypeptide (V
65+10), two chains (65KDa and 10KDa) of otch polypeptide link by the interchain disulphide bridges.The protein kinase A (PKA) that the utilization thrombocyte discharges is being tieed up out the Ser of that booth
378Specific phosphorylation reaction (korc-Grodzicki et al., the 1988a at place; Korc-Grodzicki et al., 1988b; Chain et al., 1990:Korc-Grodzicki et al., 1990; Chain et al., 1991a), we see just that recently the blood plasma enzyme is at Arg
361-Ser
362Key has cut specifically ties up out Na Ting, and this key is positioned at apart from causing this albumen to produce two chain form (V
65+10) 18 amino acid places, upstream of endogenous cutting part.We have also reported the result of blood plasma enzyme cutting, the avidity of tieing up out between that booth and PAI-1 obviously reduces (Chain et al., 1991b), and fixedly tie up out that booth and excited this cutting, find that this dimension that helps in the matrix goes out that booth cutting in the glycosaminoglycan of ECM.Fig. 1 has described our mechanism based on this discovery imagination, and by this mechanism, the blood plasma endonuclease capable presses down the generation of resistance it self by transmitting feedback signal.
In the Fibrinolytic initial stage, the plasminogen incitant is converted into the blood plasma enzyme with plasminogen.This is possible, is fixed (catching) because PAI-1 ties up out that booth molecule then, and tieing up out that booth molecule imagination is in ECM(Pollanen et al., 1988 by the glycosaminoglycan predetermined fixed).PAI-1's is fixing by stoping its arrival and suppressing the plasminogen incitant and this inhibitor has been reduced in the part.When the level of blood plasma enzyme was too high, excessive blood plasma enzyme was then preferably blocked the dimension that is fixed in subendothelium and is gone out that booth molecule.The result, fixed PAI-1 is transferred with balance between the PAI-1 that separates (moving), like this, in blood plasma, be converted into solvable (moving) thus the dimension PAI-1 that goes out the release of that booth molecule then can arrive and suppress the generation that the plasminogen incitant stops the blood plasma enzyme.In fact this release representing the change position, is fixed in PAI-1 that ECM-goes out that booth in conjunction with the dimension of (the blood plasma enzyme is clamped) and changes the soluble dimension that does not fetter for the blood plasma enzyme into and go out that booth molecule, and this has shown the height affinity to PAI-1.
The destruction of the Regulation Mechanism that the blood plasma enzyme is produced is widely used in clinical.The discovery of several aspects shows by natural incitant (as tPA, streptokinase, urokinase) and the activation of plasminogen has been constituted the many purposes biological means that is used for through blood plasma enzyme action protein dissolving barrier.No matter having under the situation of blood clot, be accidental that form or no longer need, and their dissolving has recovered free unobstructed and dynamic blood flow.Yet (Danoet al., 1985 are shifted in infiltration tissue and formation; Mignatti et al., 1989; Cajot et al., 1990) malignant cell, neurocyte in the neurotization and the blood vessel in the vasculogenesis (Mignatti et al., 1989) have obviously utilized very identical mechanism.In inflammation, ovulation, organization restructuring and development, also show and comprised plasminogen/blood plasma enzyme system.
Based on the different viewpoint of function, obviously plasmin activity must be in strict adjusting control down with the effect that guarantees partial, a site-restriction and limit its duration of service.
PAI-1 plays an important role in the activation of regulating plasminogen, and so, it also relates to the dissolving of regulating biological barrier by the blood plasma enzyme.The result, by with the functional interaction of PAI-1, tie up out Na Ting and relate to fibrinolysis, inflammation, ovulation, organization restructuring and development, vasculogenesis, neurotization, pernicious and tumour cell invasion procedure (Dano et al., 1985, Mignatti et al., 1989, Cajot et al., 1990).
Some come from conjunction with the peptide class of tieing up out the heparin in Na Ting site also shown recently mediation tie up out Na Ting-zymoplasm-antithrombin mixture and human endothelial cell to combine (de Boer et al., 1992) disclosed peptide corresponding sequence be A
341-R
355, K
348-R
361, R
357-R
370, H
366-R
379, and N
371-L
383These identical peptides also are used for determining heparin, PAI-1 and the plasminogen binding site (Kost et al., 1992) in the carboxyl terminal part of tieing up out that booth in drawing research.These publications are not described or suggestion is used for medicinal use with these peptides.
The present invention has been found that now from tieing up out the biological activity that the synthetic Toplink of that booth molecule deutero-is regulated PAI-1.Like this, these peptides can stop PAI-1 to arrive and suppress the plasminogen incitant, and perhaps they can allow PAI-1 to arrive and suppress the plasminogen incitant.
Peptide of the present invention (at this, being set at the BP peptide) is made up of aminoacid sequence, and this sequence is whole or in part with corresponding corresponding to the primary amino acid sequence of tieing up out that booth molecule 348-380 position.
Therefore the present invention also relates to and contains the pharmaceutical composition of synthetic peptide as active constituent, and this synthetic peptide contains and comprises that partly or entirely the dimension with following molecular formula goes out the amino-acid residue K of that booth
348-A
380Sequence:
348
KKQRFRHRNRKGYRSQRGHSRGRNQNSRR
380
PSRA
With its salt, functional derivatives and analogue, described analogue obtains by one or more aminoacid replacement, increase or disappearance, like this, such peptide bulk property relevant with its high positive charge density and overall water-wet behavior kept, above-mentioned peptide and its salt, functional derivatives and analogue thereof can be regulated the biological activity of PAI-1, and said composition also contains pharmaceutically acceptable carrier.
The invention still further relates to as tieing up out that booth amino-acid residue K comprising of above-mentioned qualification
348-A
380The synthetic peptide of part or all of sequence and its salt, functional derivatives and analogue thereof, but do not comprise peptide A
341-R
355, K
348-R
361, R
357-R
370, H
366-R
379, and N
371-L
383
In this paper table I and II, provided the illustrative embodiment of peptide of the present invention.The number of amino-acid residue used herein is meant that the dimension of being inferred in 1985 by Jenne and Stanley goes out the sequence of that booth.First sequence description of table in the I tie up out the K of Na Ting (VN)
348-W
382Sequence.These peptides can contain whole optically active L configurations or D configuration or wherein part for the L configuration, other is the amino acid of D configuration (underscoring is D configuration amino-acid residue in the table II).
The table I
The table II
348 363
BP6 K K Q R F R H R N R K G Y R S Q
BP7 K K Q R F R H R N R K G Y R S Q
BP8 K K Q R F R H R N R K G Y R S Q
BP14 N R K G Y R S Q
The peptide of prevention PAI-1 arrival and inhibition plasminogen incitant will be promoted the plasminogen activation and be the blood plasma enzyme, and cause fibrinolysis.This is that a class contains and comprises the K that ties up out that booth
348-R
370The position is K most preferably
348-Q
363The peptide of all or part of sequence of position, they will be suitable for use as the factor solvating agent, be used for the treatment of myocardial infarction, deep-vein thrombosis forms as leg venous thrombosis, disseminated inravascular coagulation, pulmonary infarction and other diseases.They can be used separately with enhancement endogenous protein dissolution process, or co-administered with plasminogen incitant (as tPA, streptokinase or urokinase).
Like this, in preferred concrete scheme, peptide of the present invention will contain and comprise the K that ties up out that booth
348-370The all or part of sequence of position, for example, this paper is called BP5(K
358-R
370) and BP6(K
348-Q
363) peptide.In this sequence, preferred concrete scheme is: this peptide contains the optically active amino-acid residue of part, or most preferably all is D configuration optically active amino acids residue, and for example this paper is called the peptide of BP7, BP8 and BP14.Peptide BP7 has and BP6(K
348-Q
363) identical sequence, but the residue of underscoring has substituted the L configuration with the D configuration in the table II.Peptide BP8 with BP6 identical sequence is arranged but its whole optically active amino acids is the D configuration.Peptide BP14(N
358-Q
363) contain the C-end parts of BP8, and its whole optically active amino acids are the D configuration.Peptide with part or all of D type optically active amino acids will be resisted the cutting action of enzyme more strongly or fully by the proteolytic enzyme that hemostasis and fibrinolysis process relate to.
Allow the peptide of PAI-1 arrival and inhibition plasminogen incitant to stop by plasminogen generation blood plasma enzyme.This class peptide contains and comprises and tie up out that booth S
362-A
380The all or part of sequence of position, it will be suitable for use as the antifibrinolysis agent, be used for the treatment of as in hemorrhagic diseases with excessive (out of control) proteolysis diseases associated that causes by the blood plasma enzyme.An example of this type of antifibrinolysis agent is the BP4 peptide.
Like this, another preferred concrete scheme is: peptide of the present invention contains and comprises the S that ties up out that booth
362-A
380The all or part of sequence of position, for example, this paper is called BP4(S
362-A
380), BP4-1(S
362-A
370) and BP4-2(S
362-A
368).
Peptide of the present invention and pharmaceutical composition will be applicable to that treatment relates to the disease of regulating PAI-1, for example, myocardial infarction, deep-vein thrombosis form as leg venous thrombosis, pulmonary infarction, disseminated inravascular coagulation, tumour cell intrusion and transfer, inflammation such as pancreatitis, hepatopathy, bacillary blood infection, gestosis and the relevant pathological symptom of proteolysis (for example in the tumor migration process) that mediates with the regulation and control of vasculogenesis or neurotization or excessive tPA.Brief description of drawings.
The molecule activity precedence diagram of Fig. 1 for relating in the display fibers protein dissolution, wherein the blood plasma enzyme can stop it self generation by the transmission of feedback information, discovery is tieed up out that booth matrix from fixed and has been discharged PAI-1 in extracellular matrix, and it comes across hemostasis place.PA, the plasminogen incitant; PAI-1, plasminogen incitant inhibitor-1; Vn ties up out Na Ting; Vn ', the dimension of blood plasma enzyme constraint goes out Na Ting; (s), dissolved; (i), insoluble; +, excite;-, suppress.
Fig. 2 goes out partial sequence (residue 348-380) figure of that booth for the dimension that shows the basic aminoacids sequence that contains positive charge, and the phosphorylation site (Ser of PKA has been described
378), endogenous cleavage site (Arg
379-Ala
380), blood plasma enzyme cleavage site (Arg
361-Ser
362) and heparin binding site (348-359 position) between relation.
Fig. 3 A-D shown from corresponding to the BP series synthetic peptide of tieing up out that booth 348-380 position to tieing up out the influence that Na Ting is incorporated into fixed PAI-1.A group-peptide BP1(square), application of sample (garden circle) BP2(triangle) and not; B group-BP3(square), application of sample (garden circle) BP4(triangle) and not; C group-BP(4-1) (square), BP(4-2) (triangle) and not application of sample (garden circle); D group-BP(4-1) (square), BP5(triangle), application of sample (hollow garden circle) the solid garden of BP6(circle) and not.A plurality of points among the figure are represented the mean value of measuring three times.Final peptide concentration is 25 μ M in the culturing mixt.
The peptide BP4-1(triangle of volumetric molar concentrations (25 μ M) such as Fig. 4 A-B has shown) with the BP5(square) (A group) and the solid garden of peptide BP5(circle) and the solid garden of BP6(enclose) (B group) tie up out that booth and fixed PAI-1 bonded relative potency in obstruction.Application of sample not: empty circle.
Fig. 5 A-C has shown the BP4(A group of different concns), the BP5(B group) and the BP6(C group) to tieing up out the influence of that booth and fixed PAI-1 bonded.A organizes-2.5 μ M(short side pieces), the empty triangle of 5 μ M(), the solid garden of 12 μ M(circle) and, 25 μ M BP4(closed squares), application of sample (empty circle) not; B organizes-2.5 μ M(short side pieces), the empty triangle of 10 μ M(), the solid garden of 25 μ M(circle) and, 100 μ m(closed squares), the solid triangle of 250 μ m BP5() and, application of sample (empty circle) not; C organizes-2.5 μ M(short side pieces), the empty triangle of 5 μ M(), the solid garden of 10 μ M(circle) and, 25 μ M BP6(closed squares), application of sample (empty circle) not.
Fig. 6 has shown that lower concentration BP6 is to tieing up out the influence of that booth and fixed PAI-1 bonded.Application of sample not: empty circle, 0.1 μ M BP6(triangle), 0.5 μ M BP6(square).
Fig. 7 has illustrated the empty circle of BP4(), BP5(short side piece) enclose with the solid garden of BP6() hinder and tie up out that booth contacts effect with PAI-1 comparison.The final concentration of tieing up out that booth is constant (0.5 μ g/ml), and the concentration of peptide changes as indicated.
Fig. 8 A-C has shown the peptide BP7(A group that contains the amino acid whose different concns of D configuration), the BP8(B group) and BP14(C organize) to tieing up out the influence that Na Ting is incorporated into fixed PAI-1.A organizes-1 μ M(short side piece), the empty triangle of 5 μ M(), the solid garden of 10 μ M BP7(circle) and, application of sample (empty circle) not; B organizes-1 μ M(short side piece), the empty triangle of 5 μ M(), the solid garden of 10 μ M BP8(circle) and, application of sample (empty circle) not; C organizes-1 μ M(short side piece), the empty triangle of 5 μ M(), the solid garden of 10 μ M BP14(circle) and, application of sample (empty circle) not.
Fig. 9 has shown the factor lytic activity of whole D type peptide BP8, measures the promotion that fibrin clot dissolves the detection fibers protein dissolution by forming with the D-dimer.
Figure 10 A-E has described the active experiment of factor of estimating BP8, and this activity is by with behind the 50 μ g/Kg dosed administrations, is estimated by the recovery of the blood pressure of beating of mouse.A group-contrast, the animal that no blood blocks; B organizes-has the animal that blood flow blocks; The animal that C group-BP-8 handled was handled back 13 minutes; The animal that D group-BP-8 handled was handled back 15 minutes; The animal that E group-BP-8 handled was handled back 18 minutes.
The description of preferred embodiment.
Peptide of the present invention has the height avidity to PAI-1, and can replace this inhibitor from tieing up out that booth.They can discharge PAI-1 with the form (being incorporated into the soluble Na Ting that ties up out) that suppresses from subendothelium or the hematoblastic surface of accumulative, thereby blocking-up blood plasma enzyme generates and fibrinolysis, or discharge PAI-1 from tieing up out that booth, make it change a potential (inactivation) form into, thereby stop inhibition and promotion fibrinolysis the plasminogen incitant.
In the external method of passing through for example dimeric formation of scleroproein D-and in culture, discharging PAI-1, or can estimate such peptide by the recovery of pulsatile flow in the animal body in vivo and regulate Fibrinolytic effect by endotheliocyte.Also can use other clinical blood that often uses tests, comprise in whole blood thrombolysis time, clot retraction and the culture of measuring thrombin time, euglobulin lysis time, dilution discharging tPA through endotheliocyte.
" salt " of term peptide used herein and its functional derivatives and analogue thereof comprises free carboxy and the salt of organic or inorganic alkali and the acid salt of free amine group of amino-acid residue.Term " functional derivatives " comprise free-OH ,-NH
2-and-the COOH radical derivative, ester for example, as the phosphoric acid ester of S, T and Y residue, ether, amine etc., term " analogue " comprises the peptide that replacement, increase or disappearance by amino acid moiety obtain, and comprises cyclic peptide.The present invention includes the salt of this peptide, functional derivatives and analogue are as long as they can regulate the biological activity of PAI-1.
By requirement of the present invention, total height positive charge density of peptide and water-wet behavior are deserved keeping, and are added into like this or the amino acid that is used for replacing the residue of tieing up out that booth molecule should require to adapt therewith.Should avoid the amino acid (E, D) of negative charge and excessive high hydrophobicity amino acid (W, F, Y, L, I).Recommend to use the amino acid (R, K, H) of positive charge.Like this, for example, the exchange between R, K and H residue is possible.
Pharmaceutical composition of the present invention contains peptide of the present invention, its functional derivatives, analogue or its salt, and pharmaceutically acceptable carrier.Any suitable composition administering mode be can use, oral, topical, injection or intravenous injection comprised.Multiple vehicle can be provided, and for example common salt solution, glucose or other carriers contain or do not contain other therapeutant.Dosage rate can change according to patient's situation or the special treatment of carrying out.For example can dissolve the blood clot of health different zones, comprise brain, deep layer vein, coronary artery etc.
When composition contained factor solvability peptide, they can be used separately or with the plasminogen incitant, organized plasminogen incitant (tPA), urokinase or streptokinase as natural or reorganization.
Use known method of pharmacy and process to prepare and be used for dosage form of the present invention.They can be pulvis or liquid suspension.
The invention still further relates to use to contain and comprise the amino-acid residue K that ties up out that booth
348-A
380The synthetic peptide of part or all of sequence, with its salt, functional derivatives and analogue thereof come the application of pharmaceutical compositions, said analogue is by one or more amino acid whose replacements, increase or disappearance obtain, like this, about the gross properties of this peptide of high positive charge density and overall water-wet behavior is kept, said peptide and salt, its functional derivatives and analogue thereof can be regulated the biological activity of PAI-1, the drug prepared composition is used for the treatment of various diseases, comprise Acute Myocardial Infarction, deep-vein thrombosis forms, pulmonary infarction, disseminated inravascular coagulation, tumour cell is invaded and is shifted, inflammation, hepatopathy, bacillary blood infection, gestosis, with with the regulation and control of vasculogenesis, or neurotization, or the relevant pathological symptom of proteolysis of excessive tPA-mediation, shift as (tumour).
In the further concrete scheme, the present invention relates to patient's methods of treatment, the disease that patient suffered from be selected from Acute Myocardial Infarction, deep-vein thrombosis formation, pulmonary infarction, disseminated inravascular coagulation, tumour cell intrusion and transfer, inflammation, hepatopathy, bacillary blood infection, gestosis with the regulation and control of vasculogenesis or neurotization or the excessive relevant pathological symptom of proteolysis of tPA-mediation, said methods of treatment comprises the peptide as active ingredient in pharmaceutical of the present invention defined herein to this type of patient's administering therapeutic significant quantity.
Following indefiniteness embodiment will the present invention will be described.
Embodiment
Materials and methods
A) buy material
From Tel-Hashomer Medical Center, Ramat-Gan, Israel buy FF human plasma.Exempting from anti-people ties up out that booth polyclonal antibody and is obtained by Calbiochem.Albumin A and o-nitrophenyl-β-D-galactopyranoside (ONPG) that beta-galactosidase enzymes connects are buied from Amersham U.K..PAI-1 is from American Diagnostica, and New York obtains.All the other chemical are the available highest product of commercial source.
B) tie up out Na Ting
Tie up out Na Ting by FF human plasma by the purification process preparation that people such as Korc-Grodzicki (1990) describe.This preparation method obtains this albumen of two kinds of molecular form usually: strand V
75Form and proteolysis are cut folder, with the double-stranded V of disulfide bridge connects
65+10Form (Dah/back and Podack, 1985; Jenne and Stanley, 1985).
C) method of described in document, carrying out
Use Bradford(1976) method described measures protein concentration.Induce by people's such as Salonen (1989) description and to tie up out Na Ting and be attached on the fixed PAI-1.Description as people such as Katagiri (1988) activates PAI-1 with sodium lauryl sulphate.With the 475A(Applied Biosystem 475A of applying biological system) the sequencer aminoacid sequence that is ranked.
Synthesizing of embodiment 1. peptides
Peptide of the present invention can make by chemosynthesis or genetic engineering technique with method well known in the art.
(Chemalog, South Plainfield N.J.) go up and use solid phase methodology (Barany and Merrified, 1980) to carry out the peptide synthetic operation on mechanical shaking table in polystyrene-1% divinylbenzene resin of chloromethylation.(t-BOC) protects alpha-amino group with uncle-butoxy carbonyl.Side-chain radical is protected with following protecting group: Serine (S) and Threonine (T) are with benzyl protection: with 2,6-dichloro benzyl protection tyrosine (Y) is with 2-benzyloxycarbonylchloride base protection Methionin (K); Protect arginine (R) with p-toluenesulfonyl: with carbobenzoxy-(Cbz) protection Histidine (H).Should synthetic coupling begin with C-terminal amino acid and resin.With the protected amino acid and the N of 3 times of molar excess, N '-dicyclohexylcarbodiimide and I-hydroxybenzotriazole etc. molar mixture be that coupling reagent carries out peptide and extends (coupling).Carry out deprotection and peptide is separated from polymer support with anhydrous HF.Use linear gradient (0.1% trifluoroacetic acid (TFA) aqueous solution is to the 75% acetonitrile/water solution of 0.1%TFA) through preparation HPLC(Lichrosorb RP-8; 7 μ m; 250 * 10mm:Merck, Darmstadt, Germany) the crude product peptide being made with extra care is pure product.
For describing PAI-1 at the binding site of tieing up out on that booth, synthesized a series of peptides, the structure of such peptide is derived from around its blood plasma enzyme cleavage site (R
361-S
362) aminoacid sequence (Chain etc., 1991b).Above show the sequence that I and II have provided prepared peptide.By measuring the structure that its amino acid whose composition and sequence have confirmed every kind of peptide.Table 1 has been represented the amino acid analysis of peptide BP4, BP4-1, BP5, BP6 and BP7.Table 2 has shown the sequential analysis of peptide BP4, BP5 and BP6.
Table 1
The amino acid of main peptide is formed in the BP series
*
Peptide A R N Q G H K F P S Y
BP4?0.85?5.32?1.98?2.14?2.0?0.98?-?-?1.0?2.4?-
(1)?(6)?(2)?(2)?(2)?(1)?(1)?(4)
BP4-1?-?2.95?-?1.06?2.0?1.12?-?-?-?1.32?-
(3)?(1)?(2)?(1)?(2)
BP5?-?4.1?-?1.10?3.0?1.02?0.86?-?-?1.56?1.05
(4)?(1)?(3)?(1)?(1)?(2)?(1)
BP6?-?5.0?1.02?2.18?1.0?0.97?3.38?0.90?-?0.81?1.10
(5)?(1)?(2)?(1)?(1)?(3)?(1)?(1)?(1)
BP7?-?5.18?0.99?2.11?1.10?0.84?2.86?1.0?-?0.86?0.92
(5)?(1)?(2)?(1)?(1)?(3)?(1)?(1)?(1)
* the value of providing is to analyze the nmole number that obtains, and the integer of expecting in the correct structure of the value representation in the bracket.
Table 2
The sequential analysis of BP4, BP5 and BP6
BP4 BP5 BP6
The ring sequence number. sequencing ring sequence number. sequencing ring sequence number. sequencing
Residue (yield
*) residue (yield
*) residue (yield
*)
1 S (58) 1 K (64) 1 K (62)
2 Q (97) 2 G (47) 2 K (67)
3 R ** 3 Y (61) 3 Q (68)
4 G (48) 4 R ** 4 R (13)
5 H (45) 5 S (20) 5 F (69)
6 S (37) 6 Q (44) 6 R (19)
7 R ** 7 R ** 7 H (14)
8 G (34) 8 G (26) 8 R (22)
9 R ** 9 H (16) 9 N (52)
10 N (28) 10 S (8) 10 R (24)
11 Q (20) 11 R ** 11 K (40)
12 N (15) 12 G (16) 12 G (34)
13 S (15) 13 R ** 13 Y (44)
14 R ** 14 R (24)
15 R ** 15 S (13)
16 P (25) 16 Q (17)
17 S (9)
18 R **
19 A (12)
* the pmoles of amino acid phenyl thiohydantoin in the derivative of Hui Shouing
* qualitative detection (to quantitative assay, yield is low excessively)
The synthetic peptide of embodiment 2.BP series is incorporated into influence on the fixed PAI-1 to tieing up out Na Ting
Be incorporated into fixedly being at war with property of the ability elisa assay of PAI-1 by every kind of synthetic peptide is suppressed to tie up out Na Ting, the synthetic peptide of every kind shown in the test chart 1 with contain the amino acid whose every kind of peptide of D configuration (BP7, BP8 and BP14).Peptide with same molar ratio (25 μ M) screens (Fig. 3) first.Consider variability possible in the test (main) because of some variability among the activated PAI-1 that applies on the culture plate.Go out that booth contrast as a reference on every culture plate side with independent dimension.
Various synthetic peptides (being 50 μ M) are dissolved in the damping fluid of 50 μ l, and this damping fluid is by PBS, Tween20(0.01%), polyoxyethylene glycol (molecular-weight average is 8KDa) (4%W/V) forms.Every duplicate samples is added to is coated with SDS-activated PAI-1(as preparation as described in (1988) such as Katagiri) the microtitration pond in.After 30 minutes, the dimension in same buffer that adds equal portions (50 μ l) in each pond goes out that booth solution (with different concns) 22 ℃ of cultivations.Cultivated 2 hours at 37 ℃ subsequently.With the specific albumin A of exempting from anti-people Vn antibody (1: 1000 diluent), connecting with beta-galactosidase enzymes then, and use adjacent nitrobenzyl-β-D-galactopyranoside (ONPG) to measure the amount that bonded is tieed up out that booth as substrate.Carry out enzymatic reaction and measure the absorbancy at 405+450nm place.Fig. 3 has shown the result.Each point among the figure is represented the mean value of measuring three times.Final peptide concentration is 25 μ M and has indicated the concentration of tieing up out that booth in the culturing mixt.
As shown in Figure 3, all are included in blood plasma enzyme cleavage site (R
361-S
362) and endogenous cleavage site (R
379-A
380) between dimension go out peptide BP1, BP2, BP3 and the BP4(A of that booth sequence and B group) suppressed to tie up out combining of that booth and PAI-1 to a certain extent.This inhibition mainly is the N-end parts owing to BP4, because BP4-1(S
362-R
370) be a kind of suitable effective inhibitors (Fig. 3, C group), and BP1(N
371-A
380) even restraining effect is arranged, also only demonstrate more weak restraining effect (Fig. 3, A group).It should be noted and only lack two amino acid whose BP4-2(S than BP4-1
362-R
368) be a kind of not too effective inhibitors.(Fig. 3, C group) shows G
367And/or R
370May contain an important biological recognition element.Further, the restraint of different sorts peptide can not be attributable simply to their positive charge, because it is found that BP4-1(net charge+4) than BP3(net charge+6) or BP4(net charge+7) more effective (Fig. 3, B and C group).
It is found that peptide BP5(K
358-R
370) more more effective than BP4-1 as inhibitor, BP5 comprises whole BP4-1 and further extends to blood plasma enzyme cleavage site (Fig. 3, D group).Further, peptide BP6(K
348-Q
363) it seems be this a series of in the most effectively (Fig. 3, D group).
Estimate the relative inhibition ability of peptide BP4, BP5 and BP6 by measuring inhibiting concentration dependent then.
As shown in Figure 4, waiting under the molar conditions, (every kind of peptide 25 μ M), BP5 has surpassed the BP4-1(A group), and BP6 is than BP5 more effective (B group).
As shown in Figure 5, three kinds of peptides of BP4, BP5 and BP6 have all obtained maximum restraining effect with the concentration increase.Then, when BP4 had maximum restraining effect (Fig. 5, A group) under 25 μ M concentration, BP5 then assigned maximum the inhibition (Fig. 5, B group) in 100 μ M concentration, and peptide BP6 has just reached maximum efficiency (Fig. 5, C group) when 2.5 μ M concentration.
This is further specified in Fig. 6, with concentration is 0.1 μ M(0.15 μ g/ml) peptide BP6 just obtained maximum PAI-1 bonded restraining effect, and in Fig. 7, compared the relative inhibition effect that goes out these peptides under the peptide concentration that booth concentration (0.5 μ g/ml) power increases in certain dimension.Can find out obviously that from this figure whole three kinds of peptides have the restraining effect (about 65%) of identical level.Yet BP6 obtains the inhibiting concentration ratio BP4 of this level or BP5 hangs down 200～500 times.
The above results shows: when some high affinity key element of PAI-1 binding site residue is positioned at K
348-R
357During the zone, complete PAI-1 binding site comprises K
348-R
270Sequence.The position of this PAI-1 binding site can explain our previous discovery (Chain etc., 1991b), i.e. the upper cutting of blood plasma enzyme R
361-S
362Key, the affinity of tieing up out between that booth and the PAI-1 weakens, and has discharged supressor, because the PAI-1 binding site is divided into two parts by this incision.
About containing the amino acid whose peptide of D configuration, Fig. 8 has shown three kinds of peptide BP7(A groups), the BP8(B group) and BP14(C organize) all be incorporated on the fixed PAI-1, but it seems that BP8 than BP7 and BP14 more firm combination is arranged.
The synthetic peptide of embodiment 3.BP series is to regulating Fibrinolytic influence
Usually use clinical blood to test and estimate synthetic peptide of the present invention Fibrinolytic regulating effect, mainly form with scleroproein D-dimer and in culture endotheliocyte discharge PAI-1 and regulate the fibrin clot dissolving.Other operable tests comprise measures thrombin time, euglobulin lysis time, the dilution whole blood thrombolysis time, and the tPA through endotheliocyte in clot retraction and the culture discharges.
3.1 form the dissolving of adjusting fibrin clot by the D-dimer
Add or do not add under the situation of tPA peptide for mensuration to fibrin clot dissolved regulating power, end user's Fibrinogen (containing some plasminogens) and zymoplasm are at the people's who is inoculated in dense layer endotheliocyte (obtaining from people's umbilical cord) generation fibrin clot glue.Like this, applying in advance or do not adding in the 24 hole tissue culturing plates of extracellular matrix with half amalgamation mode cultivator endotheliocyte (HUVEC).Washed cell, and the 0.4ml fibrin gel is superimposed on the cell, this fibrin gel by 3mg/ml human fibrinogen and 1 unit zymoplasm mix (Kabi, Sweden).Scleroproein was condensed 1 hour at 37 ℃, and the 0.4ml substratum (M-199 that contains 10% foetal calf serum (FCS)) that will contain test peptides is added on the fibrin gel.To add or not add the tPA dilution of testing material in the supernatant liquor.And cultivated 6 hours with cell.The sample of supernatant liquor is taken from test on D-dimer EIA.Use derives from Agen Biomedical Ltd, and (Brisbane, medicine box Austrilia) is by the D-dimer content in people such as Rylatt (1983) the enzyme immunoassay test supernatant liquor.Be with or without IPA(0.09U/ml) when existing, after cultivating 6 hours, measure the dimeric formation of D-in two pond supernatant liquors with test peptides.At the back 1 hour different peptide solution of adding in fibrin gel that condenses.Under the test conditions that does not add peptide and use, such grumeleuse dissolved in the time of about 24 hours, and this is because plasminogen is activated by the tPA that cell discharges.Also can add exogenous tPA to quicken this process and to simulate the tPA therapeutic action.Table 3 has been listed this result.
Table 3
The biological activity of peptide is regulated fibrin clot dissolved ability measurement by it
Peptide final concentration (M) adds tPA and generates survey by the D-dimer
The clot dissolution (%) of amount
*
(on average)
BP4 1.5×10
-9+ 76
4.5×10
-9+ 57
1.5×10
-8+ 48
4.5×10
-6+ 60
BP5 4.5×10
-9+ 183
1.5×10
-8+ 174
4.5×10
-7+ 144
4.5×10
-6+ 132
1×10
-6- 191
1×10
-8- 135
1×10
-6- 187
* adopting contrast is 100%.Therefore, surpass 100% value and show and promoted fibrinolysis, show and weakened fibrinous dissolving and be lower than 100% value.
Measured value surpass 100% factor dissolving peptide for example BP5 and BP6 quickened this process, measured value be lower than 100% antifibrinolysis peptide for example BP4 then suppress this process.
In the above-mentioned peptide of mentioning, BP6 is the most effectively factor dissolving peptide of being tested, and finds out this another campaign that can carry out from the BP6 with different concns.Table 4 has been summarized this result.
Table 4
The biological activity of peptide BP6 is regulated fibrin clot dissolved ability measurement by it
The final concentration of BP6 (M) is given birth to the test number by the D-dimer
The clot dissolution that becomes to measure
(%)
*(on average)
1×10
-5145 4
1×10
-6162 4
3.3×10
-7163 4
1×10
-7120 8
3.3×10
-8147 4
1×10
-8168 2
3.3×10
-9125 2
* adopting contrast is 100%
Value greater than 100% shows the promotion fibrinolysis.
Shown in this paper Fig. 3～7, it is consistent that this result and BP6 tie up out the relative capacity that Na Ting is incorporated on the fixed PAI-1 with respect to the obstruction of other peptide.
In further testing, measured full D configuration peptide BP8(K
348-Q
363) the factor lytic activity.Repeat six times with identical blood plasma storehouse (taking from healthy donor) and carry out (hexaplicates) these tests.The blood plasma of Citrated is covered on people's the endotheliocyte, and condenses with the 1U/ml zymoplasm.Trier is diluted in the HBSS(Hank ' s balanced salt solution that 250 μ l contain 1%FCS) in and be covered on the plasma clot.Sampling is used for the D-dimer assay after 6 hours.As shown in Figure 9, BP8 obviously promotes fibrinolysis, compares (this promoter action) with control group (ultra-Right end does not add peptide) and reaches 2.5 times.
3.2 animal body inner fibrin dissolved is influenced
This peptide is estimated to animal test injection peptide animal (as mouse, rabbit, cavy) body inner fibrin dissolved influence.This test system comprises the plasminogen in the experiment with measuring blood plasma, anti-blood plasma enzyme of α-2 and the D-dimer that adds or do not add IPA.In this system, tPA can inject with peptide, in order to measure them to Fibrinolytic additive regulating effect.
Use following test method to estimate the solute effect of the factor of BP8 in mouse.
3.2.a animal.From Harlan Olac, U.K. buys male Wistar SPF mouse, and makes it adapt to Sackler School of Medicine, (Tel-Aviv, Animal House Isreal).Used animal is the adult rats at 5-6 monthly age, and weight is 400～500 grams.
3.2.b the preparation of animal.Blood pressure and the heart rate of the clear-headed mouse of test.With tribromoethyl alcohol (2,2, in the 2-methyl-2-butanols of 2-tribromoethyl alcohol in being diluted in salt solution) slight anesthesia test animal, and the conduit of implanting extended immobilization at the caudal artery place is used for the measurement of blood pressure, and the conduit of implanting extended immobilization at the jugular vein place is used for intravenously administrable.Make animal operative results and anesthesia, tested later in 24 hours at implantation catheter at least.Implantation catheter is in order to test the blood pressure of mouse in the mouse cage, and does not need other any processing and operations.The salt solution (500 units per ml) of heparinization is filled with in blood coagulation in conduit when avoiding spending the night, and in salt solution (50 units per ml) flushing of duration of test with heparinization.
3.2.c blood pressure and heart rate record and data analysis.Ductus arteriosus is connected with the Statham blood pressure transducer, and on Graass multipath polygraph recording blood pressure.Sampling and the analysis of ibm compatible personal computer machine and make its digitizing from the signal of polygraph record with the DAS8A/D transmodulator.By carrying out data processing in 10 seconds in the sampling of the frequency place of 250Hz.Detection by the peak is analyzed signal.This process has been taken passages systolic pressure, diastolic pressure and mean blood pressure, and the heart rate that preestablishes the timed interval.
3.2.d administration.Although impose heparin after the conduit ligamentopexis, ductus arteriosus condensed in 3-4 days after surgery, injected fibrinolysis peptide BP8 this moment.Give these mouse through the ductus arteriosus injecting normal saline, but blood is flowed out, and do not have pulse and blood pressure recording.Recover in the ability of pulsation blood pressure recording and the ductus arteriosus the effusive difficulty or ease of blood by this medicine and estimate its effect.The drug level that the intra-arterial administration enters ductus arteriosus is 50 μ g/Kg.
Figure 10 has shown this type of result's a embodiment, and wherein A group and B group have been described healthy mouse (contrast does not have blood flow to block) respectively and had blood flow to block the pulsation blood pressure of mouse.After afterwards BP8 treatment, can see that (E group) blood pressure of pulsing after 18 minutes obviously recovers.
3.3 discharge PAI-1 through endotheliocyte in the substratum
The method of pressing people such as Chmielewske (1983) is measured the PAI-1 in the supernatant liquor of HUVEC cell.In brief, with half amalgamation mode (adding or do not add ECM) culturing cell.Substratum is changed into the Hank ' s balanced salt solution that contains 2%FCS.And the substances that will add or not add blood plasma enzyme (5 μ g/ml) is diluted in this damping fluid and with cell cultivated 6 hours.The PAI-1 activity of the sample of cell conditioned medium liquid is taken from analysis.
For analyzing PAI-1, with cell conditioned medium liquid with quantitative tPA(40U/ml) cultivate, and measure the residual activity of tPA by substrate, this substrate by plasminogen (0.1 μ g/ml), produce color substance (S-2251) and stimulator (Kabi, Sweden) composition.After substrate is cultivated, measure O.D. at the 405nm place and calculate the content of inhibitor through calibration curve.Also the medicine box of available purchase is measured PAI-1 antigen.
BP6 and BP4 with different concns test.Table 5 and 6 has been summarized this result.
Table 5
The BP6 that measures in model human endothelial cell system is to the active shadow of PAI-1
BP6 final concentration (M) PAl activity (is lower than the test number
The % of contrast)
*(on average)
1×10
-519.5 8
1×10
-636.5 8
3.3×10
-739 5
1×10
-719 2
1×10
-819 2
* the contrast of Cai Yonging is 100%.
Table 6
The BP4 that measures in model human endothelial cell system is to the active influence of PAI-1
BP4 final concentration (M) PAl activity (exceeds the test number
According to (%)
*(on average)
1×10
-529 3
1×10
-647 3
3.3×10
-717 3
1×10
-70 2
* the contrast of Cai Yonging is 100%.
The factor dissolving effect (table 5) of BP6 is to express by its active ability that reduces PAI-1 in the human endothelial cell system, so just, improve the active of plasminogen incitant (tPA) and caused the increase and the accelerating fibers protein dissolution of plasmin activity, and the antifibrinolysis effect (table 6) of BP4 is to express by its active ability of PAI-1 that improves in the same system, has so just reduced the active of plasminogen incitant (tPA) and has caused plasmin activity to reduce and the fibrinolysis of slowing down.
3.4 euglobulin lysis time
With cold acetate (0.016M) dilution citric acid blood plasma, and precipitated by centrifugation after 10 minutes, precipitation is dissolved in borate buffer solution, and by using CaCl
2(0.025M) calcification is condensed it again.Cultivate this grumeleuse down at 37 ℃.Generally grumeleuse dissolves during 2～3 hours.Before settling step, substances is added in the blood plasma.
3.5 the whole blood thrombolysis time of dilution
With the dilution proportion 1ml whole blood of veronal buffer with 1: 10.Add zymoplasm (40U/ml) the formation thrombus that 100 μ l cultivated.The blood that solidifies was cultivated 48 hours at 37 ℃.Usually blood clot will dissolve or exist after 48 hours.The adding of factor solvating agent will be shortened the dissolution of blood clot time.
Reference
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Claims (25)
1, contains and comprise corresponding to dimension and go out that booth K with following formula
348-A
380The synthetic peptide of the amino acid whose part or all of sequence of position:
348
KKQRFRHRNRKGYRSQRGHSRGRNQNSRR
380
PSRA
Salt with this peptide, its functional derivatives and analogue thereof, above-mentioned analogue can obtain by one or more amino acid whose replacements, increase or disappearance, like this, just kept this peptide with its high positive charge density and the overall relevant bulk property of water-wet behavior, above-mentioned peptide and its salt, functional derivatives and analogue thereof can be regulated plasminogen incitant inhibitor-1 biological activity of (claiming PAl-l behind this paper), but do not comprise peptide A
341-R
355
K
348-R
361，R
357-R
370，H
366
-R
379, and N
371-L
383
2, according to the peptide of claim 1, by stoping PAI-1 to arrive and suppressing the biological activity that the plasminogen incitant is regulated PAI-1.
3, according to the peptide of claim 1, by PAI-1 being arrived and suppressing the biological activity that the plasminogen incitant is regulated PAI-1.
4, according to any one peptide in the claim 1 to 3, wherein all amino-acid residues have the L configuration.
5, according to the peptide of claim 4, be called BP4 at this, its aminoacid sequence is corresponding to the S that ties up out that booth
362-A
380The position
6, according to the peptide of claim 4, be called BP4-1 at this, its aminoacid sequence is corresponding to the S that ties up out that booth
362-R
370The position.
7, according to the peptide of claim 4, be called BP5 at this, its aminoacid sequence is corresponding to the K that ties up out that booth
358-R
370The position.
8, according to the peptide of claim 4, be called BP6 at this, its aminoacid sequence is corresponding to the K that ties up out that booth
348-Q
263The position
9, according to any one peptide in the claim 1 to 3, wherein some amino-acid residue has the D configuration.
10, according to the peptide of claim 9, be called BP7 at this, its aminoacid sequence is corresponding to the K that ties up out that booth
348-Q
363The position, and the amino acid of line below has the D configuration:
348 363
KKQRFRHRNRKGYRSQ
11, according to one peptide any in the claim 1 to 3, wherein whole optically active amino acid have the D configuration.
12, according to the peptide of claim 11, be called BP8 at this, its aminoacid sequence is corresponding to the K that ties up out that booth
348-Q
363The position
13, according to the peptide of claim 11, be called BP14 at this, its aminoacid sequence is corresponding to the N that ties up out that booth
356-Q
363The position
14, be used to prepare the purposes of factor dissolved drug composition according to the peptide of claim 1 or 2.
15, according to claim 14, corresponding to tieing up out that booth K
348-R
370The purposes of the peptide of position.
16, according to any one peptide in the claim 7,8,10,12 or 13 according to the purposes of claim 15.
17, be used to prepare the purposes of antifibrinolytic pharmaceutical composition according to the peptide of claim 1 or 3.
18, according to claim 17, corresponding to tieing up out that booth S
362-A
380The purposes of the peptide of position.
19, according to any one peptide in claim 5 or 6 according to the purposes of claim 17.
20, be used to prepare the treatment and the excessive purposes of the pharmaceutical composition of fibrinolysis diseases associated such as hemorrhagic diseases according to claim 17 to 19.
21, the purposes that is used for pharmaceutical compositions according to the peptide of claim 1, this pharmaceutical composition is used for the treatment of Acute Myocardial Infarction, deep-vein thrombosis formation, pulmonary infarction, disseminated inravascular coagulation, tumour cell intrusion and migration, inflammation, hepatopathy, bacillary blood infection, gestosis, and with the regulation and control of vasculogenesis or neurotization or the excessive relevant pathological symptom of proteolysis of tPA mediation.
22, drug regimen, this pharmaceutical composition contains synthetic peptide, and this synthetic peptide contains and comprises corresponding to the dimension with following formula and go out that booth K
348-A
380The amino acid whose part or all of sequence of position:
348
KKQRFRHRNRKGYRSQRGHSRGRNQNSRR
380
PSRA
This pharmaceutical composition contains the salt of this synthetic peptide, functional derivatives and analogue thereof, above-mentioned analogue can obtain by one or more amino acid whose replacements, increase or disappearance, so just, kept this peptide with its high positive charge density and the relevant bulk property of overall water-wet behavior, above-mentioned peptide and its salt, functional derivatives and analogue thereof can be regulated the biological activity of PAI-1, and this medicine contains pharmaceutically acceptable carrier.
23, according to the pharmaceutical composition of claim 22, said composition is used for the treatment of Acute Myocardial Infarction, deep-vein thrombosis formation, pulmonary infarction, disseminated inravascular coagulation, tumour cell intrusion and migration, inflammation, hepatopathy, bacillary blood infection, gestosis, with the regulation and control of vasculogenesis or neurotization or the excessive relevant pathological symptom of proteolysis of tPA mediation.
24, according to the factor dissolved substance composition of claim 22.
25, according to the antifibrinolytics compositions of claim 22.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IL10268892A IL102688A (en) | 1992-07-30 | 1992-07-30 | Synthetic peptides derived from vitronectin and pharmaceutical compositions comprising them |
| IL102688 | 1992-07-30 | ||
| IL104296 | 1992-12-31 | ||
| IL104296A IL104296A0 (en) | 1992-12-31 | 1992-12-31 | Synthetic peptides and pharmaceutical compositions comprising them |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1091746A true CN1091746A (en) | 1994-09-07 |
Family
ID=26322490
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN93116860A Pending CN1091746A (en) | 1992-07-30 | 1993-07-30 | The synthetic peptide of derived from vitronectin and the pharmaceutical composition that contains these synthetic peptides |
Country Status (8)
| Country | Link |
|---|---|
| US (1) | US5491129A (en) |
| EP (1) | EP0589181A3 (en) |
| JP (1) | JPH06184196A (en) |
| KR (1) | KR940005671A (en) |
| CN (1) | CN1091746A (en) |
| AU (1) | AU4436393A (en) |
| CA (1) | CA2101614A1 (en) |
| HU (1) | HUT64977A (en) |
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| AU2403995A (en) * | 1994-05-10 | 1995-11-29 | Hamilton Civic Hospitals Research Development, Inc. | Methods and compositions to enhance endogenous fibrinolytic activity |
| DE69629826T2 (en) | 1995-10-23 | 2004-07-01 | Children's Medical Center Corp., Boston | THERAPEUTIC ANTIANGIOGENIC COMPOSITIONS AND METHODS |
| NZ330537A (en) * | 1995-12-13 | 2001-06-29 | Childrens Medical Center | Kringle 5 rerion of plasminogen as an endothelial cell proliferation inhibitor |
| US20020022588A1 (en) * | 1998-06-23 | 2002-02-21 | James Wilkie | Methods and compositions for sealing tissue leaks |
| AU2713500A (en) | 1998-12-23 | 2000-07-31 | G.D. Searle & Co. | Method of using a matrix metalloproteinase inhibitor and one or more antineoplastic agents as a combination therapy in the treatment of neoplasia |
| US7101989B1 (en) | 1999-07-09 | 2006-09-05 | University Of North Carolina At Chapel Hill | DsrA protein and polynucleotides encoding the same |
| EP1130031A1 (en) * | 2000-02-25 | 2001-09-05 | Universitair Medisch Centrum Utrecht | Method for inhibiting angiogenesis using molecules that enhance plasmin formation or prolong plasmin activity |
| US20040009920A1 (en) * | 2000-12-04 | 2004-01-15 | Erkki Ruoslahti | Methods and compositions for inhibiting tumor growth and angiogenesis |
| US20070003552A1 (en) * | 2002-07-09 | 2007-01-04 | Gebbink Martijn F B | Cross-beta structure comprising amyloid binding proteins and methods for detection of the cross-beta structure, for modulating cross-beta structures fibril formation and for modulating cross-beta structure-mediated toxicity and method for interfering with blood coagulation |
| EP1380290A1 (en) | 2002-07-09 | 2004-01-14 | Universitair Medisch Centrum Utrecht | Cross-beta structure pathway and its therapeutic relevance |
| US20090202980A1 (en) * | 2005-03-21 | 2009-08-13 | Crossbeta Biosciences B.V. | Cross-Beta Structure Comprising Amyloid Binding Proteins and Methods for Detection of the Cross-Beta Structure, for Modulating Cross-Beta Structures Fibril Formation and for Modulating Cross-Beta Structure-Mediated Toxicity and Method for Interfering With Blood Coagulation |
| US20080118529A1 (en) * | 2005-07-13 | 2008-05-22 | Gebbink Martijn Frans Ben Gera | Adjuvation Through Cross -Beta Structure |
| EP1910844B1 (en) * | 2005-07-13 | 2012-04-18 | Crossbeta Biosciences B.V. | Cross-beta structure binding compounds |
| US8114832B2 (en) * | 2005-07-13 | 2012-02-14 | Crossbeta Biosciences B.V. | Method for detecting and/or removing a protein comprising a cross-beta structure from a pharmaceutical composition |
| EP2058001A1 (en) * | 2007-11-08 | 2009-05-13 | Crossbeta Biosciences B.V. | Enhancement of immunogenicity of antigens |
| EP2058000A1 (en) * | 2007-11-08 | 2009-05-13 | Crossbeta Biosciences B.V. | Immunogenic compositions capable of activating T cells |
| EP3004181B1 (en) * | 2013-05-27 | 2019-01-02 | Agency For Science, Technology And Research | Heparan sulphate |
| JP7093954B2 (en) * | 2019-07-11 | 2022-07-01 | 公立大学法人福井県立大学 | PAI-1 inhibitor and composition containing mackerel-derived peptide as an active ingredient |
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|---|---|---|---|---|
| JP2864152B2 (en) * | 1990-06-20 | 1999-03-03 | 寶酒造株式会社 | Functional polypeptide |
-
1993
- 1993-07-29 US US08/098,005 patent/US5491129A/en not_active Expired - Fee Related
- 1993-07-29 HU HU9302217A patent/HUT64977A/en unknown
- 1993-07-29 EP EP93112173A patent/EP0589181A3/en not_active Withdrawn
- 1993-07-29 CA CA002101614A patent/CA2101614A1/en not_active Abandoned
- 1993-07-30 KR KR1019930014746A patent/KR940005671A/en not_active Application Discontinuation
- 1993-07-30 JP JP5227772A patent/JPH06184196A/en active Pending
- 1993-07-30 AU AU44363/93A patent/AU4436393A/en not_active Abandoned
- 1993-07-30 CN CN93116860A patent/CN1091746A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| AU4436393A (en) | 1994-02-03 |
| HU9302217D0 (en) | 1993-10-28 |
| EP0589181A3 (en) | 1995-02-22 |
| KR940005671A (en) | 1994-03-22 |
| HUT64977A (en) | 1994-03-28 |
| CA2101614A1 (en) | 1994-01-31 |
| US5491129A (en) | 1996-02-13 |
| JPH06184196A (en) | 1994-07-05 |
| EP0589181A2 (en) | 1994-03-30 |
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